Images were acquired using an Olympus BX53 fluorescence microscope, Leica MZ10F fluorescence stereomicroscope, or Carl Zeiss 700/780 laser scanning confocal microscope. All Sox2–EGFP images were taken using an Olympus BX53 fluorescence microscope under the same settings and the same exposure times for signal intensity comparison. Images were analyzed using Photoshop CC 2015 or ImageJ software (version 1.53).
Bx53 fluorescence microscope
Manufactured by Leica
Sourced in Germany
The BX53 is a fluorescence microscope designed and manufactured by Leica. It is a high-performance microscope capable of advanced imaging and analysis of fluorescent samples. The BX53 utilizes specialized optics and illumination systems to enable detailed observation and study of fluorescently labeled specimens.
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4 protocols using bx53 fluorescence microscope
1
Fluorescence Microscopy Image Analysis
2
Chromosome-specific FISH Analysis
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The sequences of 5S rDNA, 45S rDNA, RsCent1, RsCent2, BrCent1, BrCent2, RsSTRa, RsSTRb, BrSTRa, BrSTRb and telomere were used as probes (Table S3 ). The probes were labeled by nick translation with different fluorochromes. Root mitotic chromosome spreads and FISH procedures were performed according to a previous method (Waminal & Kim, 2012 ). For directly labeled probes, slides were immediately used for FISH after fixation with 4% paraformaldehyde, without subsequent pepsin and RNase pretreatment. Images were captured with an Olympus BX53 fluorescence microscope equipped with a Leica DFC365 FS CCD camera and processed using Cytovision v.7.2 (Leica Microsystems, Wetzlar, Germany).
3
Fluorescent In Situ Hybridization of Ginseng Chromosomes
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Root mitotic chromosome spreads were prepared from stratified seeds provided by the Korea P. ginseng Corporation Natural Resources Research Institute (Daejeon, Korea), according to the methods described by Waminal et al [24] (link). Pg167TR and B. oleracea 5S rDNA amplicons were labeled with Texas Red-5-dUTP (NEL417001EA; Perkin Elmer, Waltham, MA, USA), and a 9-kb fragment of 45S rDNA (18S–5.8S–25S) [30] (link) was labeled with Alexa Fluor 488-5-dUTP (C11397; Invitrogen, Waltham, MA, USA) through direct nick-translation labeling. FISH procedures were performed as described previously [24] (link). Briefly, slides were immediately used for FISH after fixation with 4% paraformaldehyde, without pepsin and RNase pretreatment. Images were captured with an Olympus BX53 fluorescence microscope equipped with a Leica DFC365 FS CCD camera, and processed using Cytovision version 7.2 (Leica Microsystems, Wetzlar, Germany). Further image enhancements and creation of the idiogram were performed in Adobe Photoshop CC.
4
Retrotransposon Mapping on Panaax Chromosomes
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Root mitotic chromosomes were prepared using the method previously described (Waminal et al., 2012 ). The retrotransposon probes were labelled directly or indirectly through nick translation of PCR product for each retrotransposon subfamilies. For direct labelling, either Texas Red‐5‐dUTP (NEL417001EA, Perkin Elmer, Waltham, USA) or Alexa Fluor 488‐5‐dUTP (C11397, Invitrogen, Eugene, USA) was directly incorporated into the DNA probes. For indirect labelling, either digoxigenin‐11‐dUTP (11‐745‐816‐910, Roche, Mannheim, Germany) or biotin (11‐745‐824‐910, Roche, Mannheim, Germany) was incorporated into the DNA probes and detection with anti‐DIG‐FITC (11‐207‐741‐910, Roche, Mannheim, Germany) or streptavidin‐Cy3 (S6402, Sigma, Deisenhofen, Germany), respectively. Chromosome numbers of P. ginseng are based on previous studies (Waminal et al., 2012 ; Waminal et al. 2016 ). Images were captured with an Olympus BX53 fluorescence microscope equipped with a Leica DFC365 FS CCD camera and processed using Cytovision ver. 7.2 (Leica Microsystems, Wetzlar, Germany). We performed further image enhancements using Adobe Photoshop CC.
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